Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with beta-D-galactosidase from Escherichia coli.
A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with beta-D-galactosidase from Escherichia coli. Using the anti-ferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 microliter of serum was 0.23-4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 microliter of serum. The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9-8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92 X(EIA) + 3.0 and 0.99 (n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of 125I-labelled anti-ferritin IgG used was not sufficiently high.
D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D005293:Ferritins / Q000097:blood* / Q000276:immunology; D005696:Galactosidases; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007074:Immunoglobulin G; D011863:Radioimmunoassay; D001616:beta-Galactosidase
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D005293:Ferritins; D005696:Galactosidases; D001616:beta-Galactosidase